Bottom Line:
These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels.CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix.We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

ABSTRACTMuch remains to be learned about the physiologic events that promote monocytes to become lymph-homing dendritic cells (DCs). In a model of transendothelial trafficking, some monocytes become DCs in response to endogenous signals. These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels. Here we show that the subpopulation of monocytes that expresses CD16 (Fcgamma receptor III) is predisposed to become migratory DCs. The vast majority of cells derived from CD16(+) monocytes reverse transmigrated, and their presence was associated with migratory cells expressing high levels of CD86 and human histocompatibility leukocyte antigen (HLA)-DR, and robust capacity to induce allogeneic T cell proliferation. A minority of CD16(-) monocytes reverse transmigrated, and these cells stimulated T cell proliferation less efficiently. CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix. The cell surface phenotype and migratory characteristics of CD16(+) monocytes were inducible in CD16(-) monocytes by preincubation with TGFbeta1. We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

fig5: Viability of reverse-transmigrated cells in the presence or absence of functional CD16. The viability of reverse-transmigrated cells was assessed by trypan blue staining after recovery from cultures lacking or containing zymosan. (A) Viability assessment from cultures in which CD16-depleted monocytes (CD16−) or the full fraction of monocytes containing both CD16+ and CD16− subpopulations (CD16mix) were applied to the endothelium. (B) Recovery of live CD16mix monocytes in the presence of neutralizing anti-CD16 mAb 3G8 was calculated in relation to the number of live cells recovered in the presence of isotype-matched control antibody UPC10 under each condition.

Mentions:
Although the above studies failed to show a functional role for CD16 in migration, we observed that zymosan stimulation after magnetic depletion of CD16+ monocytes led to a decreased viability in the reverse-transmigrated population (Fig. 5 A). However, viability was uniformly high in the absence of this maturation stimulus. To determine whether survival was functionally related to expression of CD16, we conducted experiments in which CD16mix monocytes were added to endothelial cultures with or without inclusion of zymosan in the collagen matrix. In some wells, monocytes were treated with neutralizing anti-CD16 mAb. In zymosan-containing cultures, the yield of live reverse-transmigrated DCs was reduced by 75% in the presence of anti-CD16 mAb (Fig. 5 B). These data suggest that stimulation of monocytes with this microbial particulate leads to cell death in the absence of functional CD16. Anti- CD16 mAb did not prevent phagocytic uptake of zymosan (unpublished data).

fig5: Viability of reverse-transmigrated cells in the presence or absence of functional CD16. The viability of reverse-transmigrated cells was assessed by trypan blue staining after recovery from cultures lacking or containing zymosan. (A) Viability assessment from cultures in which CD16-depleted monocytes (CD16−) or the full fraction of monocytes containing both CD16+ and CD16− subpopulations (CD16mix) were applied to the endothelium. (B) Recovery of live CD16mix monocytes in the presence of neutralizing anti-CD16 mAb 3G8 was calculated in relation to the number of live cells recovered in the presence of isotype-matched control antibody UPC10 under each condition.

Mentions:
Although the above studies failed to show a functional role for CD16 in migration, we observed that zymosan stimulation after magnetic depletion of CD16+ monocytes led to a decreased viability in the reverse-transmigrated population (Fig. 5 A). However, viability was uniformly high in the absence of this maturation stimulus. To determine whether survival was functionally related to expression of CD16, we conducted experiments in which CD16mix monocytes were added to endothelial cultures with or without inclusion of zymosan in the collagen matrix. In some wells, monocytes were treated with neutralizing anti-CD16 mAb. In zymosan-containing cultures, the yield of live reverse-transmigrated DCs was reduced by 75% in the presence of anti-CD16 mAb (Fig. 5 B). These data suggest that stimulation of monocytes with this microbial particulate leads to cell death in the absence of functional CD16. Anti- CD16 mAb did not prevent phagocytic uptake of zymosan (unpublished data).

Bottom Line:
These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels.CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix.We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

ABSTRACTMuch remains to be learned about the physiologic events that promote monocytes to become lymph-homing dendritic cells (DCs). In a model of transendothelial trafficking, some monocytes become DCs in response to endogenous signals. These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels. Here we show that the subpopulation of monocytes that expresses CD16 (Fcgamma receptor III) is predisposed to become migratory DCs. The vast majority of cells derived from CD16(+) monocytes reverse transmigrated, and their presence was associated with migratory cells expressing high levels of CD86 and human histocompatibility leukocyte antigen (HLA)-DR, and robust capacity to induce allogeneic T cell proliferation. A minority of CD16(-) monocytes reverse transmigrated, and these cells stimulated T cell proliferation less efficiently. CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix. The cell surface phenotype and migratory characteristics of CD16(+) monocytes were inducible in CD16(-) monocytes by preincubation with TGFbeta1. We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.